Astrocytes play a central role in maintaining synaptic homeostasis, regulating neurotransmitter clearance, and supporting neuronal function, all of which are integral to cognition. In Alzheimer's brain, astrocytic dysfunction contributes significantly to the rapid progression of the disease pathology. Aβ deposition induces oxidative alterations in the astrocytic glutamate transporter EAAT2, leading to impaired glutamate uptake and elevated extracellular glutamate levels, which drive excitotoxicity. This disruption adversely impacts synaptic plasticity, neuronal survival, and cognitive integrity, highlighting the pivotal role of astrocytes in the early phases of AD. Restoration of EAAT2 function is critical to mitigating disease progression and preserving cognitive function. Through in silico analyses, three potential allosteric modulators of EAAT2 were identified. These compounds were further tested on Aβ-intoxicated primary neuronal-astrocyte mixed cultures, with their efficacy evaluated via immunoblotting for EAAT2 expression, in vitro glutamate assays for functional assessment, and morphological analyses of astrocytes and neurons using GFAP and β-III tubulin markers. Notably, PTID and NDHP significantly enhanced EAAT2 expression and function, reducing extracellular glutamate levels and ameliorating excitotoxic damage. Moreover, these compounds demonstrated neuroprotective effects by promoting dendritic growth and branching in β-III tubulin-positive neurons. Immunocytochemical analyses further revealed an increased number of β-III tubulin and GFAP-positive cells, suggesting that PTID and NDHP not only improved neuronal integrity but also supported astrocytic resilience and functionality. The findings of the current study underscore the critical role of astrocytes in glutamate homeostasis and cognition, showcasing the therapeutic potential of EAAT2 allosteric modulators, particularly PTID and NDHP, in alleviating Aβ-induced neurotoxicity. By enhancing EAAT2 function, these modulators restore astrocytic support for synaptic health, offering a promising avenue for addressing cognitive decline in AD.
